- Email: [email protected]
The Efficacy of an Ultrapure Alginate Gel in Reducing Adhesion Formation in a Rat Model of Blood Contamination
j o u r n a l o f s u r g i c a l r e s e a r c h s e p t e m b e r 2 0 1 9 ( 2 4 1 ) 2 7 1 e2 7 6
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
The Efficacy of an Ultrapure Alginate Gel in Reducing Adhesion Formation in a Rat Model of Blood Contamination Jeroen C. Hol, MD,* Chema Strik, MD, PhD, Ankit A. Chaturvedi, MSc, PhD, Roger M.L.M. Lomme, BSc, Harry van Goor, MD, PhD, FRCS, Martijn W.J. Stommel, MD, PhD, and Richard P.G. ten Broek, MD, PhD Department of surgery, Radboud University Medical Center, Nijmegen, The Netherlands
article info
abstract
Article history:
Background: Formation of peritoneal adhesions is the most frequent complication of abdominal
Received 3 December 2018
and pelvic surgery and comprises a lifelong risk of adhesion-related morbidity and mortality.
Received in revised form
Some of the existing antiadhesive barriers are less effective in the presence of blood. In this
12 March 2019
study, we investigate the efficacy and safety of ultrapure alginate gel in the presence of blood.
Accepted 22 March 2019
Methods: In experiment 1 (30 rats), 1 mL ultrapure alginate gel was compared with no
Available online 28 April 2019
intervention in a model of cecal abrasion and persisting peritoneal bleeding by incision of the epigastric artery. In experiment 2 (30 rats), 2 mL ultrapure alginate gel was compared
Keywords:
with no intervention in a model where a 1 mL blood clot was instilled intra-abdominally
Ultrapure alginate gel
and a cecal resection was performed. The primary endpoint was the incidence and
Adhesion barrier
severity of adhesions after 14 d.
Abdominal adhesions
Results: In experiment 1, seven of 15 rats in the experimental group had intra-abdominal adhesions compared with 13 of 15 rats in the control group (P ¼ 0.05); 3 of 15 rats had adhesions at the site of injury compared with 12 of 15 rats in the control group (P < 0.01). The severity and extent of adhesions was also reduced (P < 0.01). In experiment 2, 12 of 13 rats had adhesions compared with 13 of 14 rats in the control group (P ¼ 1.00). Conclusions: Ultrapure alginate gel reduces the incidence and severity of adhesion in the presence of persisting bleeding, but not in a model of cecal resection and blood clot. ª 2019 Elsevier Inc. All rights reserved.
Introduction Formation of intraperitoneal adhesions is the most frequent cause of long-term complications after abdominal and pelvic surgery. Intestinal obstruction, female infertility, and chronic abdominal pain are known adhesion-related complications after abdominal surgery.1 Furthermore, adhesiolysis during
repeat surgeries results in an increase of operative time and risk of iatrogenic organ injuries such as an enterotomy.2 Adhesiolysis is associated with more postoperative complications and readmissions.2 Despite the burden of intraperitoneal adhesions, adhesion barriers are seldom applied. Existing adhesion barriers have important drawbacks limiting their clinical use.3 In the past
* Corresponding author. Radboud University Nijmegen Medical Center, Department of Surgery, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands. Tel.: þ31243613808; fax: þ31243635115. E-mail address: [email protected] (J.C. Hol). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.03.035
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decades, there has been an increase in the use of laparoscopy.4 Although the use of laparoscopy reduces the incidence of adhesions, adhesions are still an important issue in laparoscopic surgery.5 Therefore, a barrier should be applicable in minimally invasive surgery. Hyaluronate carboxymethylcellulose (Seprafilm, Genzyme Corporation, Cambridge, MA) forms a brittle sticky sheet which makes it difficult to apply during laparoscopy.6 Liquid barriers with low viscosity, such as icodextrin 4% (Adept, Baxter, Deerfield, IL), give broad coverage of the abdominal cavity but may be less effective at sites with severe surgical injury.7 Oxidized regenerated cellulose (ORC, Interceed, Johnson & Johnson, New Brunswick, NJ) has a reduced efficacy and can have an adverse aggravating effect on adhesion formation in the presence of blood.8,9 ORC is a procoagulant that can promote fibrin deposition at sites of incomplete hemostasis, resulting in adhesion formation instead of prevention.10 This has limited indications for ORC to smaller gynecological operations with little blood loss.11 Therefore, a new generation of antiadhesive barriers is needed. An ideal antiadhesive barrier should be safe and effective in preventing both adhesion formation and reformation in the presence of blood and a contaminated environment. Furthermore, it should be easy to apply in both open and laparoscopic surgery. Finally, the barrier should be able to remain in place without sutures or staples. We recently reported on a new antiadhesive barrier, ultrapure alginate gel, which showed good antiadhesive effects in preventing adhesion formation and reformation in animal experiments.12,13 Furthermore, its usage does not impair the healing of colonic anastomosis and is safe in a contaminated environment.14,15 The mucoadherent properties of the gel and the viscosity make it suitable for use in laparoscopy. Moreover, the polydextran sulfate in the gel increases its resistance to bacterial colonization.16 At present, the impact of blood contamination on the efficacy of this barrier has not been clarified. When effective in the presence of blood, ultrapure alginate gel could be superior to other barriers and have a broader indication area in major abdominal surgery where often no meticulous hemostasis can be achieved. This experimental data are considered mandatory to ensure safety before clinical use.17 Our present study aims to assess the efficacy and safety of ultrapure alginate gel in the presence of blood contamination: first in a model of persistent bleeding, mimicking clinical situation of oozing after major abdominal surgery and second, in a model of hematoma formation after cecal resection, mimicking the clinical situation of a colonic resection with a postoperative hematoma.
Methods Animals A total of 60 male outbred Wistar rats (HsdCpb:WU; Harlan, Horst, the Netherlands), 7-9 wk of age, were used in two consecutive experiments. The rats weighed 230-280 g and were housed in filter-topped cages (two rats per cage) at 22 C23 C with a 12 hour day cycle for at least 5 d before the experiment. The cages were enriched with a shelter and
nesting material and were randomly placed on the shelves. During the experimental period, rats had free access to acidified tap water and standard rodent chow (Ssniff R/M-H; BioServices B.V.) The study protocol was submitted and approved by the Animal Ethics Review Committee of the Radboud University Medical Center and performed in the GLP-certified animal research laboratory.
Randomization and allocation Rats were randomly assigned to their respective groups using a computerized program for each experiment (Excel; Microsoft). The efficacy of ultrapure alginate gel in the presence of blood was determined in two models. In experiment 1, a model of persisting peritoneal bleeding was used to mimic the clinical situation of a diffuse oozing surface. Rats were randomly allocated to a control (no intervention, n ¼ 15) and experimental group (1 mL ultrapure alginate gel, n ¼ 15). Experiment 2 assessed the efficacy of ultrapure alginate gel in the presence of a blood clot and a contaminated peritoneum. This situation simulates a bowel resection after which an intra-abdominal hemorrhage occurs. Rats were randomly allocated to either a control group (no intervention, n ¼ 15) or to an experimental group (2 mL ultrapure alginate gel, n ¼ 15). The dose in experiment 2 was higher than in experiment 1 because the area at risk of adhesions was larger due to the blood clot placed centrally in the abdomen. Sufficient gel was used in both experiments to cover the full area at risk.
Perioperative care, surgical procedure, and sacrifice Rats received 5 mg/kg carprofen (Rimadyl; Pfizer Animal Health) at least 15 min before surgery and once daily until the second day after surgery for analgesia. All rats were prepared for surgery by shaving and disinfecting the skin with 70% alcohol and an iodine solution, and anesthetized by inhalation of isoflurane (3%) in a mixture of oxygen:pressurized air (1:1). The surgical procedures were carried out by the same experienced researcher (R.L.) blinded to the group assignment, in a sterile manner, and during surgery, rats were placed on a warm plate to avoid hypothermia. All rats underwent a 4 cm midline laparotomy, which was closed in two layers, Vicryl 30 (Johnson & Johnson) was used to close the fascia/muscle, and staples were used for the skin. In experiment 1, the cecum was exteriorized and an area of 1 by 2 cm was abraded by 20 strokes with a gauze. The opposing area (1 by 2 cm) of the abdominal sidewall was injured by excising the peritoneum with scissors. One epigastric artery was cut in addition to ensure persisting peritoneal bleeding. The injured areas were placed together and fixed with two sutures (Ethilon 8-0; Johnson & Johnson). In experiment 2, the cecum was exteriorized, ligated, and excised after which a 1 mL blood clot was placed in the abdominal cavity. The clot was not fixated. This blood was drawn from the tail veins of control rats from the same breed from a previous experiment on the day of sacrifice. Blood was collected in a cup and left until a clot formed. Ultrapure alginate gel was applied to the damaged surfaces in the experimental groups by a second person (C.S. or A.C.)
273
hol et al ultrapure alginate gel and blood
after inducing the injuries or applying the blood clot. Animals were resuscitated with 2 mL isotonic sodium chloride (0.9%) solution administered subcutaneously and placed in an incubator to prevent postoperative hypothermia. No antibiotics were given. Rats were euthanized in random order in the laboratory by using carbon dioxide asphyxiation in the morning on postoperative day 14. The abdomen was opened using a U-incision and inspected for adhesion formation and abnormalities.
Outcomes The primary endpoint of both experiments was the overall incidence of adhesions; secondary endpoints included the incidence of adhesion formation at the site of injury, the severity and extent of adhesions at the site of injury, and number of abscesses. The severity of adhesions was classified macroscopically using the Zu¨hlke classification: 0, no adhesions; 1, filmy adhesions: easy to separate by blunt dissection, no vascularization; 2, mild adhesions: blunt dissection possible but partly sharp dissection required; 3, strong adhesions: lysis possible by sharp dissection only, clear vascularization; 4, very strong adhesions: lysis possible by sharp dissection only and organ injury almost unavoidable.18 The extent of adhesions was scored as 0 ¼ 0%; 1 ¼ 1%-25%, 2 ¼ 26%-50%, 3 ¼ 51%-75%, and 4 ¼ 76%-100% of the injured area covered by adhesions. All outcomes were scored in random order by an experienced researcher (R.L.), blinded to group assignment. Safety endpoints were herniation of abdominal contents through the abdominal wall and excessive weight loss (more than 20% of their initial weight.) Rats that met the criteria of a safety endpoint were euthanized. A safety analysis was performed by comparing mortality and number of animals reaching humane endpoints between the groups and between control group and ultrapure alginate geletreated animals.
group and the ultrapure alginate gel group at the start or the end of the experiment. Seven of 15 rats in the experimental group had any intraabdominal adhesions compared with 13 of 15 rats in the control group (P 0.05). At the site of injury, ultrapure alginate gel also significantly reduced the incidence of adhesions (P < 0.01); 3 of 15 rats had adhesions at the site of injury compared with 12 of 15 rats in the control group. Ultrapure alginate gel significantly reduced the number, severity, and extent of adhesions at the site of injury (P < 0.01) (Table 1). Other adhesions were located between the cecum and fat pad, omentum or small bowel or between sidewall and fat pad or omentum. No rats in the control group had abdominal fluid present at the time of sacrifice compared with 12 rats in the group treated with ultrapure alginate gel (P < 0.01).
Experiment 2: clean-contaminated peritoneum and the presence of a blood clot Three rats were excluded from the study because they lost more than 20% of their initial weight. At postmortem examination, 1 rat in the ultrapure alginate gel group showed to have a fecal peritonitis due to leakage at the cecal ligation site and 1 rat in the ultrapure alginate gel group had a postoperative ileus. One rat in the control group showed to have an adhesive bowel obstruction. There were no significant differences between the control and ultrapure alginate gel group with regard to weight at the beginning or end of the study. The overall incidence of adhesions was 12 of 13 rats in the ultrapure alginate gel group and 13 of 14 rats in the control group (P 1.00). The number of adhesions in each rat showed no statistical significant difference (P 0.94) (Table 2).
Statistical analysis For the experiments, a power calculation was performed (G*Power 3.1.2)19 using a one-sided Fisher’s exact test with a ¼ 0.05 and b ¼ 0.8. Based on experiences in previous experiments, the expected incidence of adhesion formation was set at 95% for the controls and 50% in the experimental group, yielding a group size of 13 animals. The calculated group size was increased by two animals to adjust for a loss of animals due to complications. Dichotomous or categorical variables are presented as absolute numbers and percentages. Continuous variables are shown as their absolute numbers. A chi square test was performed to assess differences in the incidence of adhesion formation. A ManneWhitney U-test was performed for continuous variables. A P-value less than 0.05 was considered significant.
Table 1 e Experiment 1: assessment of ultrapure alginate in persistent peritoneal bleeding model; severity and extent of adhesions at the site of injury. Adhesions
Adhesions cecum and sidewall
Control
1 mL ultrapure alginate gel
Significance
12/15
3/15
P < 0.01
P < 0.01
Zu¨hlke score No adhesions
3
3
1
0
1
2
3
0
3
9
1
4
0
10
No adhesions
2
2
1%-25%
0
1
26%-50%
0
1
51%-75%
1
1
76%-100%
2
0
Extent
Results Experiment 1: persistent peritoneal bleeding No rats in this experiment died or were excluded. There were no differences between the weight of the rats in the control
P < 0.01
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Two of 13 rats in the ultrapure alginate gel group showed to have a Zu¨hlke grade 3 adhesions compared with 6 rats having a total of 10 Zu¨hlke grade 3 adhesions in the control group (P 0.09). The number of Zu¨hlke 1, 2, and 4 adhesions in the animals did not differ significantly between both groups (Table 2). None of the rats in the control group had abdominal fluid present at the time of sacrifice compared with 11 rats in the group treated with ultrapure alginate gel (P < 0.01). Ten rats treated with ultrapure alginate gel had gel residue present in the abdomen. The blood clot was still present in the abdomen 14 d after the operation in 9 of 14 control rats and in 6 of 13 rats treated with ultrapure alginate gel (P 0.45). Subgroup analysis of presence of the clot versus absence of the clot showed no significant differences in incidence, number, severity, or extent of adhesions.
Discussion Ultrapure alginate gel is safe and effective for prevention of adhesion formation in the presence of persistent peritoneal bleeding. In the presence of cecal resection and an allogenic blood clot, the use of ultrapure alginate gel does not prevent adhesion formation. These experiments were performed in accordance with current standards concerning sample size, randomization, allocation concealment, and blinded outcome assessment.20,21 We chose to assess adhesion formation after 14 d as mesothelial healing is completed at that point, making the formation of new adhesions unlikely.22 All surgical procedures were carried out by one researcher and assessment of adhesions was carried out by a blinded observer in a random sequence, thereby minimizing inter- and intra-observer differences. Ultrapure alginate gel decreased the incidence of adhesion formation in a model with persisting peritoneal bleeding. The current model was comparable with the
Table 2 e Experiment 2: assessment of ultrapure alginate gel in a clean-contaminated peritoneum and the presence of a blood clot; number and severity of adhesions in rats in the group treated with ultrapure alginate gel and rats in the control group. Adhesions
Control
2 mL ultrapure alginate gel
No adhesions
1
1
1 adhesion
4
4
2 adhesions
7
5
3 or more adhesions
2
3
Significance
P ¼ 0.94
Zu¨hlke score 1
2
7
2
11
14
3
10
2
4
1
1
P ¼ 0.09
model used to assess the efficacy of ORC in the presence of blood.9 The efficacy of ORC was reduced in a model of persistent bleeding, where hemostasis could not be achieved; the reason for this, however, has not been fully elucidated.9,11 A possible explanation is found in the hemostatic properties of ORC. ORC is a procoagulant that induces fibrin formation, which is the first step in adhesion formation, thereby potentially promoting the formation of adhesions.10 Furthermore, the material is acidic and can cause necrosis of cells, acidosis secondary to blood loss or inadequate tissue perfusion, and degradation of the material requires an inflammatory response, further promoting adhesion formation.23 Ultrapure alginate gel is still effective in the presence of postoperative bleeding; therefore, its use is promising in patient undergoing major abdominal surgery, where oozing is unavoidable and has to be accepted. Ultrapure alginate gel might have some remote antiadhesive effects. Being a viscous gel instead of a liquid barrier makes migration of the gel less likely. This distant effect seems limited. In previous experiments, ultrapure alginate gel had little antiadhesive effect, when not placed at the damaged area but elsewhere in the abdomen.12 A benefit of a viscous gel is that it remains better in place in the operative areas most at risk for adhesions, creating better local efficacy. On the other hand, most rats in the first experimental group had abdominal fluid present at time of sacrifice. These fluids are possibly remnants of the gel or an effect of continued bleeding. Previous studies have shown that this higher dose is safe. If the fluid was remnant of the gel, more fluid was expected in the higher dose group in experiment 2. In some of the previous experiments with ultrapure alginate gel, histopathological examination showed no significant giant cell reaction and no clear evidence that the fluids were remnant from the gel.12 Surgery of the digestive tract in humans typically implies both a clean-contaminated operation and some postoperative hematoma formation. Our aim in experiment 2 was to mimic this situation by performing a cecal resection and adding a blood clot. The clot was placed in the center of the abdomen instead of the damaged area, where the gel is placed, mimicking the clinical scenario of laparoscopy, where absolute hemostasis distant from the damaged area often cannot be achieved. To our knowledge, no similar experiments have been performed previously in preclinical testing of antiadhesive barriers. An allogenic blood clot was used because it seemed practical to standardize the amount of blood in contact with ultrapure alginate gel in this cleancontaminated abdomen. Harvesting blood from each animal to produce an autologous would have been more elaborate and might have led to volume depletion and anemia in some of the rats. The number and severity of adhesions was greater than expected in this experiment, suggesting a possibly aggravated immune response to the allogenic blood. In a previous study, ultrapure alginate gel effectively reduced adhesions in a model with solely a cleancontaminated peritoneum due to the creation of a colonic anastomosis.14 In our current cecal resection experiment, 90% of the rats treated with ultrapure alginate gel showed to have
hol et al ultrapure alginate gel and blood
adhesions, suggesting a substantial additional adhesion promoting effect of the allogenic blood clot after a cleancontaminated operation. This might be the combined promoting effect of inflammation to contamination and the presence of an allogenic blood clot. This is supported by the fact that the blood clot was still present 14 d after the operation in more than half of the rats. The number of animals with a blood clot still present did not differ significantly between the groups in experiment 2, suggesting no competition between the gel and the blood clot. Therefore, the presence of the clot at 14 d is an unlikely explanation for the difference in adhesion formation. Clotted blood is known to have a substantial added effect to adhesion formation; preformed clots have shown to produce widespread adhesions even without peritoneal injury.24 It contains a fibrinous network on which fibroblasts may proliferate, forming adhesions.25 The Wistar rats used in this experiment are outbred rats and therefore have different genotypes, which could have triggered an additional immune response as seen in experimental allogenic transplantation research.26 Because of the possible substantial added effect of the allogenic blood clot, the model might be refined by using inbred strains or an autologous blood clot. The present study together with other recently preclinical studies by our group showed that ultrapure alginate gel has promising properties for a next-generation adhesion barrier. Being a gel makes it suitable for handling in minimally invasive surgery. The gel does not compromise anastomotic healing nor did its use increase mortality in a rat model of experimental peritonitis.14,15 The ultrapure alginate gel seems more effective than other barriers in prevention of adhesion reformation, although efficacy in preventing reformation is lower than in preventing primary de novo adhesions.13 This current experiment demonstrated that the gel remains effective in the environment of oozing, which is an important attribute for use in major abdominal surgery, where complete hemostasis can often not be achieved.
Acknowledgment Authors’ contributions: J.C.H. collected the data, performed the statistical analysis, interpreted the data, and drafted the first and subsequent versions of the manuscript. C.S. participated in the design of the experiment, participated in the execution of the experiment, collected the data, performed the statistical analysis interpreted the data, and critically revised the manuscript. A.A.C. participated in the design of the experiment, participated in the execution of the experiment, and collected the data. R.M.L.M.L. participated in the design of the experiment, participated in the execution of the experiment, and collected the data. H.v.G. supervised the experiment, participated in the design of the experiment, collected the data, interpreted the data, and critically revised the manuscript. M.W.J.S. participated in the design of the experiment and critically revised the manuscript. R.P.G.t.B. supervised the experiment, participated in the design of the experiment, interpreted the data, and critically revised the manuscript.
275
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
references
1. ten Broek RP, Issa Y, van Santebrink EJ, et al. Burden of adhesions in abdominal and pelvic surgery: systematic review and met-analysis. BMJ. 2013;347:f5588. 2. ten Broek RP, Strik C, Issa Y, Bleichrodt RP, van Goor H. Adhesiolysis-related morbidity in abdominal surgery. Ann Surg. 2013;258:98e106. 3. ten Broek RP, Stommel MW, Strik C, et al. Benefits and harms of adhesion barriers for abdominal surgery: a systematic review and meta-analysis. Lancet. 2014;383:48e59. 4. Kolfschoten NE, van Leersum NJ, Gooiker GA, et al. Successful and safe introduction of laparoscopic colorectal cancer surgery in Dutch hospitals. Ann Surg. 2013;257:916e921. 5. Stommel MW, ten Broek RP, Strik C, et al. Multicenter observational study of adhesion formation after open-and laparoscopic surgery for colorectal cancer. Ann Surg. 2017;267:743e748. 6. Lim R, Morril JM, Lynch RC, et al. Practical limitations of bioresorbable membranes in the prevention of intraabdominal adhesions. J Gastrointest Surg. 2009;13:35e41. discussion 41-42. 7. Brown CB, Luciano AA, Martin D, et al. Adept (icodextrin 4% solution) reduces adhesions after laparoscopic surgery for adhesiolysis: a double-blind, randomized, controlled study. Fertil Steril. 2007;88:1413e1426. 8. Farquhar C, Vandekreckhove P, Watson A, Vail A, Wiseman D. Barrier agents for preventing adhesions after surgery for subfertility. Cochrane Database Syst Rev. 2000:CD000475. 9. Wiseman DM, Gottlick LE, Diamond MP. Effect of thrombininduced hemostasis on the efficacy of an absorbable adhesion barrier. J Reprod Med. 1992;37:766e770. 10. Haney AF, Doty E. A barrier composed of chemically cross-linked hyaluronic acid (Incert) reduces postoperative adhesion formation. Fertil Steril. 1998;70:145e151. 11. Wiseman DM, Gottlick-Iarkowski L, Kamp L. Effect of different barriers of oxidized regenerated cellulose (ORC) on cecal and sidewall adhesions in the presence and absence of bleeding. J Invest Surg. 1999;12:141e146. 12. Chaturvedi AA, Lomme RM, Hendriks T, van Goor H. Prevention of postsurgical adhesions using an ultrapure alginate-based gel. Br J Surg. 2013;100:904e910. 13. Chaturvedi AA, Lomme RM, Hendriks T, van Goor H. Ultrapure alginate gel reduces adhesion reformation after adhesiolysis. Int J Colorectal Dis. 2014;29:1411e1416. 14. Chaturvedi AA, Lomme RM, Hendriks T, van Goor H. Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength. J Surg Res. 2014;192:432e439. 15. Chaturvedi AA, Buyne OR, Lomme RM, Hendriks T, van Goor H. Efficacy and safety of ultrapure alginate-based antiadhesion gel in experimental peritonitis. Surg Infect (Larchmt). 2015;16:410e414. 16. Contreras A, Vazquez D, Carrasco L. Inhibition, by selected antibiotics, of protein synthesis in cells growing in tissue cultures. J Antibiot (Tokyo). 1978;31:598e602. 17. DFaD A. Guidance for resorbable adhesion barrier devices for use in abdominal and/or pelvic surgery; guidance for industry. U.S: Center of Devices and Radiological health;
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2008. Available at: https://www.fda.gov/MedicalDevices/ DeviceRegulationandGuidance/GuidanceDocuments/ ucm072879.htm. Zuhlke HV, Lorenz EM, Straub EM, Savvas V. [Pathophysiology and classification of adhesions]. Langenbecks Arch Chir Suppl II Verh Dtsch Ges Chir. 1990;Suppl II:1009e1016. Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Methods. 2009;41:1149e1160. Kilkenny C, Browne WJ, Cuthi I, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Vet Clin Pathol. 2012;41:27e31. Watkins PE. Standards for the use of animals in surgical research. Br J Surg. 2013;100:580e581.
22. Holmdahl L, Risberg B, Beck DE, et al. Adhesions: pathogenesis and prevention-panel discussion and summary. Eur J Surg Suppl. 1997;577:56e62. 23. Lewis KM, Spazierer D, Urban MD, Lin L, Redl H, Goppelt A. Comparison of regenerated and non-regenerated oxidized cellulose hemostatic agents. Eur Surg. 2013;45:213e220. 24. Ryan GB, Grobety J, Majno G. Postoperative peritoneal adhesions. A study of the mechanisms. Am J Pathol. 1971;65:117e148. 25. diZerega GS, Campeau JD. Peritoneal repair and post-surgical adhesion formation. Hum Reprod Update. 2001;7:547e555. 26. Lee AD, Gama-Rodrigues J, Galvao FH, Waitzberg DL. Study of morbidity in orthotopic small intestine transplantation with Wistar rats. Experimental study. Arq Gastroenterol. 2002;39:39e47.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
The Efficacy of an Ultrapure Alginate Gel in Reducing Adhesion Formation in a Rat Model of Blood Contamination Jeroen C. Hol, MD,* Chema Strik, MD, PhD, Ankit A. Chaturvedi, MSc, PhD, Roger M.L.M. Lomme, BSc, Harry van Goor, MD, PhD, FRCS, Martijn W.J. Stommel, MD, PhD, and Richard P.G. ten Broek, MD, PhD Department of surgery, Radboud University Medical Center, Nijmegen, The Netherlands
article info
abstract
Article history:
Background: Formation of peritoneal adhesions is the most frequent complication of abdominal
Received 3 December 2018
and pelvic surgery and comprises a lifelong risk of adhesion-related morbidity and mortality.
Received in revised form
Some of the existing antiadhesive barriers are less effective in the presence of blood. In this
12 March 2019
study, we investigate the efficacy and safety of ultrapure alginate gel in the presence of blood.
Accepted 22 March 2019
Methods: In experiment 1 (30 rats), 1 mL ultrapure alginate gel was compared with no
Available online 28 April 2019
intervention in a model of cecal abrasion and persisting peritoneal bleeding by incision of the epigastric artery. In experiment 2 (30 rats), 2 mL ultrapure alginate gel was compared
Keywords:
with no intervention in a model where a 1 mL blood clot was instilled intra-abdominally
Ultrapure alginate gel
and a cecal resection was performed. The primary endpoint was the incidence and
Adhesion barrier
severity of adhesions after 14 d.
Abdominal adhesions
Results: In experiment 1, seven of 15 rats in the experimental group had intra-abdominal adhesions compared with 13 of 15 rats in the control group (P ¼ 0.05); 3 of 15 rats had adhesions at the site of injury compared with 12 of 15 rats in the control group (P < 0.01). The severity and extent of adhesions was also reduced (P < 0.01). In experiment 2, 12 of 13 rats had adhesions compared with 13 of 14 rats in the control group (P ¼ 1.00). Conclusions: Ultrapure alginate gel reduces the incidence and severity of adhesion in the presence of persisting bleeding, but not in a model of cecal resection and blood clot. ª 2019 Elsevier Inc. All rights reserved.
Introduction Formation of intraperitoneal adhesions is the most frequent cause of long-term complications after abdominal and pelvic surgery. Intestinal obstruction, female infertility, and chronic abdominal pain are known adhesion-related complications after abdominal surgery.1 Furthermore, adhesiolysis during
repeat surgeries results in an increase of operative time and risk of iatrogenic organ injuries such as an enterotomy.2 Adhesiolysis is associated with more postoperative complications and readmissions.2 Despite the burden of intraperitoneal adhesions, adhesion barriers are seldom applied. Existing adhesion barriers have important drawbacks limiting their clinical use.3 In the past
* Corresponding author. Radboud University Nijmegen Medical Center, Department of Surgery, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands. Tel.: þ31243613808; fax: þ31243635115. E-mail address: [email protected] (J.C. Hol). 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.03.035
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decades, there has been an increase in the use of laparoscopy.4 Although the use of laparoscopy reduces the incidence of adhesions, adhesions are still an important issue in laparoscopic surgery.5 Therefore, a barrier should be applicable in minimally invasive surgery. Hyaluronate carboxymethylcellulose (Seprafilm, Genzyme Corporation, Cambridge, MA) forms a brittle sticky sheet which makes it difficult to apply during laparoscopy.6 Liquid barriers with low viscosity, such as icodextrin 4% (Adept, Baxter, Deerfield, IL), give broad coverage of the abdominal cavity but may be less effective at sites with severe surgical injury.7 Oxidized regenerated cellulose (ORC, Interceed, Johnson & Johnson, New Brunswick, NJ) has a reduced efficacy and can have an adverse aggravating effect on adhesion formation in the presence of blood.8,9 ORC is a procoagulant that can promote fibrin deposition at sites of incomplete hemostasis, resulting in adhesion formation instead of prevention.10 This has limited indications for ORC to smaller gynecological operations with little blood loss.11 Therefore, a new generation of antiadhesive barriers is needed. An ideal antiadhesive barrier should be safe and effective in preventing both adhesion formation and reformation in the presence of blood and a contaminated environment. Furthermore, it should be easy to apply in both open and laparoscopic surgery. Finally, the barrier should be able to remain in place without sutures or staples. We recently reported on a new antiadhesive barrier, ultrapure alginate gel, which showed good antiadhesive effects in preventing adhesion formation and reformation in animal experiments.12,13 Furthermore, its usage does not impair the healing of colonic anastomosis and is safe in a contaminated environment.14,15 The mucoadherent properties of the gel and the viscosity make it suitable for use in laparoscopy. Moreover, the polydextran sulfate in the gel increases its resistance to bacterial colonization.16 At present, the impact of blood contamination on the efficacy of this barrier has not been clarified. When effective in the presence of blood, ultrapure alginate gel could be superior to other barriers and have a broader indication area in major abdominal surgery where often no meticulous hemostasis can be achieved. This experimental data are considered mandatory to ensure safety before clinical use.17 Our present study aims to assess the efficacy and safety of ultrapure alginate gel in the presence of blood contamination: first in a model of persistent bleeding, mimicking clinical situation of oozing after major abdominal surgery and second, in a model of hematoma formation after cecal resection, mimicking the clinical situation of a colonic resection with a postoperative hematoma.
Methods Animals A total of 60 male outbred Wistar rats (HsdCpb:WU; Harlan, Horst, the Netherlands), 7-9 wk of age, were used in two consecutive experiments. The rats weighed 230-280 g and were housed in filter-topped cages (two rats per cage) at 22 C23 C with a 12 hour day cycle for at least 5 d before the experiment. The cages were enriched with a shelter and
nesting material and were randomly placed on the shelves. During the experimental period, rats had free access to acidified tap water and standard rodent chow (Ssniff R/M-H; BioServices B.V.) The study protocol was submitted and approved by the Animal Ethics Review Committee of the Radboud University Medical Center and performed in the GLP-certified animal research laboratory.
Randomization and allocation Rats were randomly assigned to their respective groups using a computerized program for each experiment (Excel; Microsoft). The efficacy of ultrapure alginate gel in the presence of blood was determined in two models. In experiment 1, a model of persisting peritoneal bleeding was used to mimic the clinical situation of a diffuse oozing surface. Rats were randomly allocated to a control (no intervention, n ¼ 15) and experimental group (1 mL ultrapure alginate gel, n ¼ 15). Experiment 2 assessed the efficacy of ultrapure alginate gel in the presence of a blood clot and a contaminated peritoneum. This situation simulates a bowel resection after which an intra-abdominal hemorrhage occurs. Rats were randomly allocated to either a control group (no intervention, n ¼ 15) or to an experimental group (2 mL ultrapure alginate gel, n ¼ 15). The dose in experiment 2 was higher than in experiment 1 because the area at risk of adhesions was larger due to the blood clot placed centrally in the abdomen. Sufficient gel was used in both experiments to cover the full area at risk.
Perioperative care, surgical procedure, and sacrifice Rats received 5 mg/kg carprofen (Rimadyl; Pfizer Animal Health) at least 15 min before surgery and once daily until the second day after surgery for analgesia. All rats were prepared for surgery by shaving and disinfecting the skin with 70% alcohol and an iodine solution, and anesthetized by inhalation of isoflurane (3%) in a mixture of oxygen:pressurized air (1:1). The surgical procedures were carried out by the same experienced researcher (R.L.) blinded to the group assignment, in a sterile manner, and during surgery, rats were placed on a warm plate to avoid hypothermia. All rats underwent a 4 cm midline laparotomy, which was closed in two layers, Vicryl 30 (Johnson & Johnson) was used to close the fascia/muscle, and staples were used for the skin. In experiment 1, the cecum was exteriorized and an area of 1 by 2 cm was abraded by 20 strokes with a gauze. The opposing area (1 by 2 cm) of the abdominal sidewall was injured by excising the peritoneum with scissors. One epigastric artery was cut in addition to ensure persisting peritoneal bleeding. The injured areas were placed together and fixed with two sutures (Ethilon 8-0; Johnson & Johnson). In experiment 2, the cecum was exteriorized, ligated, and excised after which a 1 mL blood clot was placed in the abdominal cavity. The clot was not fixated. This blood was drawn from the tail veins of control rats from the same breed from a previous experiment on the day of sacrifice. Blood was collected in a cup and left until a clot formed. Ultrapure alginate gel was applied to the damaged surfaces in the experimental groups by a second person (C.S. or A.C.)
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after inducing the injuries or applying the blood clot. Animals were resuscitated with 2 mL isotonic sodium chloride (0.9%) solution administered subcutaneously and placed in an incubator to prevent postoperative hypothermia. No antibiotics were given. Rats were euthanized in random order in the laboratory by using carbon dioxide asphyxiation in the morning on postoperative day 14. The abdomen was opened using a U-incision and inspected for adhesion formation and abnormalities.
Outcomes The primary endpoint of both experiments was the overall incidence of adhesions; secondary endpoints included the incidence of adhesion formation at the site of injury, the severity and extent of adhesions at the site of injury, and number of abscesses. The severity of adhesions was classified macroscopically using the Zu¨hlke classification: 0, no adhesions; 1, filmy adhesions: easy to separate by blunt dissection, no vascularization; 2, mild adhesions: blunt dissection possible but partly sharp dissection required; 3, strong adhesions: lysis possible by sharp dissection only, clear vascularization; 4, very strong adhesions: lysis possible by sharp dissection only and organ injury almost unavoidable.18 The extent of adhesions was scored as 0 ¼ 0%; 1 ¼ 1%-25%, 2 ¼ 26%-50%, 3 ¼ 51%-75%, and 4 ¼ 76%-100% of the injured area covered by adhesions. All outcomes were scored in random order by an experienced researcher (R.L.), blinded to group assignment. Safety endpoints were herniation of abdominal contents through the abdominal wall and excessive weight loss (more than 20% of their initial weight.) Rats that met the criteria of a safety endpoint were euthanized. A safety analysis was performed by comparing mortality and number of animals reaching humane endpoints between the groups and between control group and ultrapure alginate geletreated animals.
group and the ultrapure alginate gel group at the start or the end of the experiment. Seven of 15 rats in the experimental group had any intraabdominal adhesions compared with 13 of 15 rats in the control group (P 0.05). At the site of injury, ultrapure alginate gel also significantly reduced the incidence of adhesions (P < 0.01); 3 of 15 rats had adhesions at the site of injury compared with 12 of 15 rats in the control group. Ultrapure alginate gel significantly reduced the number, severity, and extent of adhesions at the site of injury (P < 0.01) (Table 1). Other adhesions were located between the cecum and fat pad, omentum or small bowel or between sidewall and fat pad or omentum. No rats in the control group had abdominal fluid present at the time of sacrifice compared with 12 rats in the group treated with ultrapure alginate gel (P < 0.01).
Experiment 2: clean-contaminated peritoneum and the presence of a blood clot Three rats were excluded from the study because they lost more than 20% of their initial weight. At postmortem examination, 1 rat in the ultrapure alginate gel group showed to have a fecal peritonitis due to leakage at the cecal ligation site and 1 rat in the ultrapure alginate gel group had a postoperative ileus. One rat in the control group showed to have an adhesive bowel obstruction. There were no significant differences between the control and ultrapure alginate gel group with regard to weight at the beginning or end of the study. The overall incidence of adhesions was 12 of 13 rats in the ultrapure alginate gel group and 13 of 14 rats in the control group (P 1.00). The number of adhesions in each rat showed no statistical significant difference (P 0.94) (Table 2).
Statistical analysis For the experiments, a power calculation was performed (G*Power 3.1.2)19 using a one-sided Fisher’s exact test with a ¼ 0.05 and b ¼ 0.8. Based on experiences in previous experiments, the expected incidence of adhesion formation was set at 95% for the controls and 50% in the experimental group, yielding a group size of 13 animals. The calculated group size was increased by two animals to adjust for a loss of animals due to complications. Dichotomous or categorical variables are presented as absolute numbers and percentages. Continuous variables are shown as their absolute numbers. A chi square test was performed to assess differences in the incidence of adhesion formation. A ManneWhitney U-test was performed for continuous variables. A P-value less than 0.05 was considered significant.
Table 1 e Experiment 1: assessment of ultrapure alginate in persistent peritoneal bleeding model; severity and extent of adhesions at the site of injury. Adhesions
Adhesions cecum and sidewall
Control
1 mL ultrapure alginate gel
Significance
12/15
3/15
P < 0.01
P < 0.01
Zu¨hlke score No adhesions
3
3
1
0
1
2
3
0
3
9
1
4
0
10
No adhesions
2
2
1%-25%
0
1
26%-50%
0
1
51%-75%
1
1
76%-100%
2
0
Extent
Results Experiment 1: persistent peritoneal bleeding No rats in this experiment died or were excluded. There were no differences between the weight of the rats in the control
P < 0.01
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Two of 13 rats in the ultrapure alginate gel group showed to have a Zu¨hlke grade 3 adhesions compared with 6 rats having a total of 10 Zu¨hlke grade 3 adhesions in the control group (P 0.09). The number of Zu¨hlke 1, 2, and 4 adhesions in the animals did not differ significantly between both groups (Table 2). None of the rats in the control group had abdominal fluid present at the time of sacrifice compared with 11 rats in the group treated with ultrapure alginate gel (P < 0.01). Ten rats treated with ultrapure alginate gel had gel residue present in the abdomen. The blood clot was still present in the abdomen 14 d after the operation in 9 of 14 control rats and in 6 of 13 rats treated with ultrapure alginate gel (P 0.45). Subgroup analysis of presence of the clot versus absence of the clot showed no significant differences in incidence, number, severity, or extent of adhesions.
Discussion Ultrapure alginate gel is safe and effective for prevention of adhesion formation in the presence of persistent peritoneal bleeding. In the presence of cecal resection and an allogenic blood clot, the use of ultrapure alginate gel does not prevent adhesion formation. These experiments were performed in accordance with current standards concerning sample size, randomization, allocation concealment, and blinded outcome assessment.20,21 We chose to assess adhesion formation after 14 d as mesothelial healing is completed at that point, making the formation of new adhesions unlikely.22 All surgical procedures were carried out by one researcher and assessment of adhesions was carried out by a blinded observer in a random sequence, thereby minimizing inter- and intra-observer differences. Ultrapure alginate gel decreased the incidence of adhesion formation in a model with persisting peritoneal bleeding. The current model was comparable with the
Table 2 e Experiment 2: assessment of ultrapure alginate gel in a clean-contaminated peritoneum and the presence of a blood clot; number and severity of adhesions in rats in the group treated with ultrapure alginate gel and rats in the control group. Adhesions
Control
2 mL ultrapure alginate gel
No adhesions
1
1
1 adhesion
4
4
2 adhesions
7
5
3 or more adhesions
2
3
Significance
P ¼ 0.94
Zu¨hlke score 1
2
7
2
11
14
3
10
2
4
1
1
P ¼ 0.09
model used to assess the efficacy of ORC in the presence of blood.9 The efficacy of ORC was reduced in a model of persistent bleeding, where hemostasis could not be achieved; the reason for this, however, has not been fully elucidated.9,11 A possible explanation is found in the hemostatic properties of ORC. ORC is a procoagulant that induces fibrin formation, which is the first step in adhesion formation, thereby potentially promoting the formation of adhesions.10 Furthermore, the material is acidic and can cause necrosis of cells, acidosis secondary to blood loss or inadequate tissue perfusion, and degradation of the material requires an inflammatory response, further promoting adhesion formation.23 Ultrapure alginate gel is still effective in the presence of postoperative bleeding; therefore, its use is promising in patient undergoing major abdominal surgery, where oozing is unavoidable and has to be accepted. Ultrapure alginate gel might have some remote antiadhesive effects. Being a viscous gel instead of a liquid barrier makes migration of the gel less likely. This distant effect seems limited. In previous experiments, ultrapure alginate gel had little antiadhesive effect, when not placed at the damaged area but elsewhere in the abdomen.12 A benefit of a viscous gel is that it remains better in place in the operative areas most at risk for adhesions, creating better local efficacy. On the other hand, most rats in the first experimental group had abdominal fluid present at time of sacrifice. These fluids are possibly remnants of the gel or an effect of continued bleeding. Previous studies have shown that this higher dose is safe. If the fluid was remnant of the gel, more fluid was expected in the higher dose group in experiment 2. In some of the previous experiments with ultrapure alginate gel, histopathological examination showed no significant giant cell reaction and no clear evidence that the fluids were remnant from the gel.12 Surgery of the digestive tract in humans typically implies both a clean-contaminated operation and some postoperative hematoma formation. Our aim in experiment 2 was to mimic this situation by performing a cecal resection and adding a blood clot. The clot was placed in the center of the abdomen instead of the damaged area, where the gel is placed, mimicking the clinical scenario of laparoscopy, where absolute hemostasis distant from the damaged area often cannot be achieved. To our knowledge, no similar experiments have been performed previously in preclinical testing of antiadhesive barriers. An allogenic blood clot was used because it seemed practical to standardize the amount of blood in contact with ultrapure alginate gel in this cleancontaminated abdomen. Harvesting blood from each animal to produce an autologous would have been more elaborate and might have led to volume depletion and anemia in some of the rats. The number and severity of adhesions was greater than expected in this experiment, suggesting a possibly aggravated immune response to the allogenic blood. In a previous study, ultrapure alginate gel effectively reduced adhesions in a model with solely a cleancontaminated peritoneum due to the creation of a colonic anastomosis.14 In our current cecal resection experiment, 90% of the rats treated with ultrapure alginate gel showed to have
hol et al ultrapure alginate gel and blood
adhesions, suggesting a substantial additional adhesion promoting effect of the allogenic blood clot after a cleancontaminated operation. This might be the combined promoting effect of inflammation to contamination and the presence of an allogenic blood clot. This is supported by the fact that the blood clot was still present 14 d after the operation in more than half of the rats. The number of animals with a blood clot still present did not differ significantly between the groups in experiment 2, suggesting no competition between the gel and the blood clot. Therefore, the presence of the clot at 14 d is an unlikely explanation for the difference in adhesion formation. Clotted blood is known to have a substantial added effect to adhesion formation; preformed clots have shown to produce widespread adhesions even without peritoneal injury.24 It contains a fibrinous network on which fibroblasts may proliferate, forming adhesions.25 The Wistar rats used in this experiment are outbred rats and therefore have different genotypes, which could have triggered an additional immune response as seen in experimental allogenic transplantation research.26 Because of the possible substantial added effect of the allogenic blood clot, the model might be refined by using inbred strains or an autologous blood clot. The present study together with other recently preclinical studies by our group showed that ultrapure alginate gel has promising properties for a next-generation adhesion barrier. Being a gel makes it suitable for handling in minimally invasive surgery. The gel does not compromise anastomotic healing nor did its use increase mortality in a rat model of experimental peritonitis.14,15 The ultrapure alginate gel seems more effective than other barriers in prevention of adhesion reformation, although efficacy in preventing reformation is lower than in preventing primary de novo adhesions.13 This current experiment demonstrated that the gel remains effective in the environment of oozing, which is an important attribute for use in major abdominal surgery, where complete hemostasis can often not be achieved.
Acknowledgment Authors’ contributions: J.C.H. collected the data, performed the statistical analysis, interpreted the data, and drafted the first and subsequent versions of the manuscript. C.S. participated in the design of the experiment, participated in the execution of the experiment, collected the data, performed the statistical analysis interpreted the data, and critically revised the manuscript. A.A.C. participated in the design of the experiment, participated in the execution of the experiment, and collected the data. R.M.L.M.L. participated in the design of the experiment, participated in the execution of the experiment, and collected the data. H.v.G. supervised the experiment, participated in the design of the experiment, collected the data, interpreted the data, and critically revised the manuscript. M.W.J.S. participated in the design of the experiment and critically revised the manuscript. R.P.G.t.B. supervised the experiment, participated in the design of the experiment, interpreted the data, and critically revised the manuscript.
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Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
references
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22. Holmdahl L, Risberg B, Beck DE, et al. Adhesions: pathogenesis and prevention-panel discussion and summary. Eur J Surg Suppl. 1997;577:56e62. 23. Lewis KM, Spazierer D, Urban MD, Lin L, Redl H, Goppelt A. Comparison of regenerated and non-regenerated oxidized cellulose hemostatic agents. Eur Surg. 2013;45:213e220. 24. Ryan GB, Grobety J, Majno G. Postoperative peritoneal adhesions. A study of the mechanisms. Am J Pathol. 1971;65:117e148. 25. diZerega GS, Campeau JD. Peritoneal repair and post-surgical adhesion formation. Hum Reprod Update. 2001;7:547e555. 26. Lee AD, Gama-Rodrigues J, Galvao FH, Waitzberg DL. Study of morbidity in orthotopic small intestine transplantation with Wistar rats. Experimental study. Arq Gastroenterol. 2002;39:39e47.